A New Fusion Protein Platform For Quantitatively Measuring Activity Of Multiple Proteases

Proteases play key roles in many fundamental cellular and viral processes. Some specific proteases are frequently applied for cleaving the fusion tags used in enhancing soluble expression of recombinant protein in Escherichia coli and rapid purification by chromatography. Ii is very important to study the protease specificity and the specific inhibitors for application in biotechnology and for therapy to cure the diseases. Escherichia coli expressing system is preferentially selected for screening the engineered protease variants and specific inhibitors owing to fast growth of the host, inexpensive culture conditions and easy manipulation. Several protein substrates for monitoring and detecting various proteases have been constructed, but they can only applied for some specific proteases, and sometime, the laborious analyzing steps are essential. Therefore, we developed a new fusion protein platform for quantitatively measuring activity of multiple proteases. The activity of the target enzyme significantly decreased by the fusion tag but rescued dramatically by the specific protease. The protease activity was analyzed by the coupled assay. The results are as follows:1. We constructed several fusion proteins incorporated independently with the different protease recognition sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, See VMA1 intein, thrombin and enterokinase). The four different fusion tags (His6, GST, CBD and MBP) were designed, if necessary. The diaminopropionate ammonia-lyase (DAL) from E. coli (eDAL) and Salmonella typhimurium (sDAL) were selected as the target protein.2. The different fusion tags affecting on soluble production of the fusion proteins were investigated by SDS-PAGE and Western blot analysis. Only MBP had the most efficiently effect on enhancing soluble production of the DALs.3. The effects of some protease inhibitors on DAL activity were examined. All the inhibitors tested had barely effect on DAL activity.4. The fusion tags impacting DAL activity were identified. Different fusion tags had various inhibition of the activity of eDAL or sDAL.5. The fusion protein substrates from crude extract and in purified form were preteolyzed by TEV protease and factor Xa. Additional the corresponded protease inhibitors caused the loss of protease activity.6. Thrombin displayed the different cleavage efficiency of two recognition sequences, but it was inactivated in the presence of the inhibitor.7. Enterokinase can cleave the eDAL but not sDAL. So, the enterokinase activity was analyzed using the fusion protein for sDAL as the substrate, which was inactivated by the inhibitor.8. The MBP had more efficient on enhancing protein production of sDAL with the rhinovirus 3C cut than GST. The processing of the designed fusion protein by rhino virus 3C with or without the protease inhibitor was assayed.9. The purified fusion protein exhibited the different self-cleavage efficiency by See VMA1 intein under two designed temperatures at different reaction time.10. The SUMO fused eDAL can be cleaved by the designed SUMO protease in vitro or in E. coli. The in vivo cleavage efficiency was related to soluble yields of SUMO protease.In conclusion, our developed new fusion protein platform based on the rescued DAL activity is efficiently applied for quantitatively measuring activity of sequence-specific and conformational-specific proteases. The protein substrate for measuring intein activity is first reported. Comparing with other constructed fusion proteins as protease substrates, the designed DAL-fusion proteins are suitable for detecting activity and specificity of different proteases. The analytical method is simple and rapid.